Explanation The activity of poly-ADP-ribose polymerases (PARPs) is reversed by poly-ADP-ribose (PAR)-degrading enzymes, of which poly-ADP-ribose glycohydrolase (PARG) and ADP ribosylhydrolase 3 (ARH3) catalyze PAR-degradation in vitro and in cells. In humans and mice, four PARG isoforms result from alternative splicing of a single PARG gene. Of them, only the protein encoded by the full-length open reading frame (hPARG111 and mPARG110) localizes to the nucleus. Most PARG activity is detected in the cytosol, which is in apparent contradiction to the nuclear localization of PARP1, the most abundant and most active PARP. Knockout of the PARG gene is embryonic lethal, whereas mice that express PARG from a gene deleted in exons 2 and 3 (PARGexon2-3), which lack nuclear mPARG110 as well as the two cytosolic isoforms mPARG101 and mPARG98, are viable. This suggests a vital role of the small murine PARG isoform mPARG63. The human counterpart of mPARG63 is a 60 kDa-protein (hPARG60) that, in contrast to mice, lacks exon 5-encoded amino acids. Owing to alternative translational start sites, the transcripts encoding the small human and murine PARG isoforms were suggested to encode proteins, in which the N-terminal amino acids constitute a mitochondrial targeting sequence (MTS) that is masked in all other PARGs. We established the genetic background of a fifth human PARG isoform hPARG55, which results from a hitherto unrecognized alternative splicing event of the primary PARG transcript. In-depth localization analysis revealed hPARG55 to be the only isoform that is targeted to the mitochondrial matrix, where the presence of poly-ADP-ribose metabolism is debated. Surprisingly, hPARG55 is catalytically inactive both in vitro and in cells. The PAR-degrading activity of hPARG55 could be restored by reintroducing exon 5-encoded amino acids. These findings could be applied to hPARG60 that also lacks exon 5, but localizes to the cytosol. In wild-type mice, we identified a splicing event leading to a cytosolic 52 kDa-PARG isoform (mPARG52), which was so far only reported in the PARGexon2-3 mutant. This isoform lacks exon 4 (encoding the MTS) and a significant portion of exon 5 supporting the conclusion that the human and the murine PARG genes encode PARG isoforms with functions different from PAR-degradation. Given these findings, we investigated the role of ARH3 in PAR degradation in mitochondria. In cells from ARH3-/- mice, targeted expression of PARP1 activity in mitochondria lead to greater accumulation of PAR. Matrix-accumulated PAR in ARH3-/- cells was no longer subject to degradation. Downregulation of PARG gene expression in ARH3-/- cells did not affect the PAR-degrading activity in mitochondria. Thus, the PAR-degrading activity we previously identified within mitochondria is carried out by ARH3, suggesting a role of this enzyme in PAR metabolism.